Conjugation of protein therapeutics with polyethylene glycol (PEG) has been shown to confer important therapeutic benefits including increased serum half-life and reduced antigenicity (Kozlowski, A., et al., J. Controlled Release (2001) 72:217-224). Each ethylene oxide unit of PEG associates with two to three water molecules, which results in the molecule behaving as if it were five to ten times as large as a protein of comparable molecular weight (Kozlowski, A., supra). The clearance rate of PEGylated proteins is inversely proportional to molecular weight (Yamaoka, T., et al., J. Pharm. Sci. (1994) 83:601-606). Below a molecular weight of approximately 20,000, the molecule is cleared in the urine. Higher-molecular-weight PEG proteins are cleared more slowly in the urine and the feces (Yamaoka, T., supra). PEGylated proteins have enhanced solubility, decreased antigenicity, decreased proteolysis, and reduced rates of kidney clearance as well as enhanced selective tumor targeting.
Currently, PEGylated forms of adenosine deaminase, asparaginase, α-IFN and a growth hormone antagonist have received regulatory approval. (Maeda, H., et al. (eds.), Advances in experimental medicine and biology: polymer drugs in the clinical stage, (2003) Vol. 519, Dordrecht, The Netherlands: Kluwer Academic/Plenum Publishers). PEG-α-IFN has been approved in two forms for treatment of hepatitis C. (Kozlowski, A., supra, and Gilbert, C. W., et al., U.S. Pat. No. 5,951,974 (1999)). Patients with refractory or recurrent acute lymphoblastic leukemia (ALL) are treated with a combination of PEG-asparaginase and methotrexate, vincristine, and prednisone (Aguayo, A., et al., Cancer (1999) 86:1203-1209). Studies also show that PEG-ADA considerably strengthened the immune system in patients with adenosine deaminase (ADA) deficiency, who are vulnerable to almost any type of infection due to inhibited development of the immune system. (Pool, R., Science (1990) 248:305; and Hershfield, M. S., Clin. Immunol. Immunopathol. (1995) 76:S228-S232). While PEGylated proteins exhibit desirable therapeutic properties, methods for protein conjugation with PEG are limited by the number and distribution of sites on proteins available for conjugation.